Biomarkers for the Diagnosis and Treatment of Pancreatic Cancer

ABSTRACT

Compositions and methods which indicate an increased risk for pancreatic carcinoma are disclosed.

This application claims priority to US provisional Application,61/021,772 filed Jan. 17, 2008, the entire contents being incorporatedby reference herein.

Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described, which was madein part with funds from the National Institutes of Health, Grant Number,CA119242.

FIELD OF THE INVENTION

This invention relates to the fields of oncology and proteomic analysis.More specifically, the invention discloses biomarkers that are presentin pancreatic cyst fluid which are indicative of an increased risk forthe development of pancreatic cancer and methods of use thereof indiagnostic and prognostic assays. Also disclosed are screening assaysutilizing the biomarkers of the invention to identify agents useful forthe treatment of pancreatic cancer.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Increasing use of high resolution computerized tomography and magneticresonance imaging in clinical practice has resulted in detection of agrowing number of pancreatic cysts (1). As a result, clinicians arefrequently asked to determine the biological nature of these cysticlesions, and to make treatment recommendations accordingly. However,there are currently no diagnostic indicators that are consistentlyreliable, obtainable, and conclusive for diagnosing and risk-stratifyingpancreatic cysts. The sensitivity of pancreatic cyst fluid cytology hasbeen reported as only 27-64% in most series. Several studies havesuggested that a variety of tumor markers (e.g., CEA(2), CA 19-9, CA15-3) may distinguish mucinous from non-mucinous cystic lesions, andalso may predict whether a cyst harbors areas of malignanttransformation (3-5).

The biologic nature and histopathologic features of pancreatic cysts arevaried (3, 6). Ten to twenty percent of pancreatic cysts are neoplastic,including neoplasms which grow as cystic structures (i.e., primarycystic neoplasms of the pancreas), and solid neoplasms that haveundergone cystic degeneration. Serous cystadenomas (microcysticadenomas) account for approximately 32-39% of the primary cysticneoplasms and have very low malignant potential. Mucinous cysticneoplasms, which include mucinous cystadenomas and intraductal papillarymucinous neoplasms, are a subgroup of primary cystic neoplasms that havemalignant potential. Nomenclature describing the evolution of theselesions, from benign to malignant, is provided elsewhere (7, 8).Mucinous cystic neoplasms and intraductal papillary mucinous neoplasms(IPMNs) account for approximately 10-45% and 21-33% of primary cysticneoplasms, respectively (6, 9-11). Two subtypes of IPMN have beendescribed (1, 12), a main duct variant and a branch duct variant; thelatter may have a more indolent course. There are other less commonforms of primary cystic neoplasms of the pancreas, such as solidpseudopapillary tumors.

In the absence of reliable methods of quantifying the malignantpotential of a suspected pre-malignant cystic neoplasm of the pancreas,if existing clinical parameters suggest the presence of one such lesionin a person that is otherwise an acceptable surgical risk, partial ortotal pancreatomy may be recommended but can result in significantmorbidity and mortality (13). Alternatively, a conservative“watch-and-wait” approach (i.e., serial imaging over time) is advocatedfor some patients, but this strategy may be suboptimal due toincremental costs accrued during surveillance, and the possibility thatmalignant transformation may occur between surveillance time points.

In light of all the foregoing, it is clear that a more reliable methodfor identifying those patients at increased risk for developingpancreatic cancer is urgently needed.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method of diagnosing anincreased risk for the development of pancreatic cancer in a human testsubject is provided. An exemplary method entails isolating a pancreaticcyst fluid specimen from the subject; analyzing the fluid specimen forthe presence of at least three biomarkers associated with increased riskof pancreatic carcinoma, wherein the presence of said biomarkers isindicative of an increased risk for pancreatic cancer, said biomarkersbeing selected from the group consisting of mucin 1, mucin 2, mucin 5AC,mucin 5B, mucin 6, CEA CAM 1, CEACAM 6, CEACAM 7, CEACAM 8, S100-A6,S100-A8, S100 A9 and S100 A-11.

The risk of pancreatic cancer increases when the proteomic analysisshows an increase in several combinations of biomarkers. These are asfollows:

a) the presence of several isoforms of mucins e.g., mucin 1, 2, 5AC, 5B,and 6;b) the presence of both mucins and certain isoforms of CEA, includingCEACAM1, 6, 7, and 8; and c) mucins and CEA are variable but CEACAM8 ispresent and at least two of S100-A6, A8, A9, or A11 are present.

Also provided is a solid support comprising antibodies which areimmunospecific for the biomarkers described above. Such supports caninclude, without limitation, filters, biacore chips, ELISA plates andthe like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table providing clinical information for the pancreatic cystfluid samples tested herein. CEA (ng/mL) and Amylase (units/mL) areresults from clinical lab immuno assays. MCA=mucinous cystadenoma.Cytology categories: A—Benign: No evidence of benign mucinousepithelium, atypical cells or carcinoma. C—Atypical/suspicious cytology.

FIGS. 2A-2O provide tables showing representatives of 137 plasmaproteins distributed among the pancreatic cyst samples. The numberspresented in FIG. 2A are emPAI scores which are roughly proportional toprotein abundance. Dark (>1), medium (0.1 to 1), and light (<0.1)shading denotes relative protein abundance. No proteins were detectedfor the empty boxes. CEA=ng/mL.

FIG. 3 is a table listing the pancreatic enzyme proteins in thepancreatic cyst samples. Legends are the same as for FIG. 2.

FIG. 4 is a table listing proteomic biomarkers for pancreatic cancere.g., mucins, CEACAMs, and S100s found in the pancreatic cyst samples.Legends are the same as for FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

There are currently no diagnostic indicators that are consistentlyreliable, obtainable, and conclusive for diagnosing and risk-stratifyingpancreatic cysts. To establish more effective diagnostic biomarkers andto provide deeper understanding about the molecular profile within thesecysts, we identified and quantified about 500 cyst fluid proteins andcorrelated the findings to clinical parameters, when available.Pancreatic cyst fluids were collected by endoscopic ultrasound-guidedfine needle aspiration (EUS-FNA) from 20 patients. The proteins in thecyst fluids were ascertained by LC/MS/MS analysis of every gel slicefrom 1 D gel fractionation of each sample, using partial peptidesequencing on a highly accurate and stable mass spectrometer.Measurements of traditional markers of mucins, amylase, and CEA wereobtained simultaneously from 15 micrograms of protein from less than 40microliters of cyst fluids per sample. The proteomic techniques utilizedprovided comprehensive information about pancreatic enzymes, plasmainfiltrate proteins, and proteins that may have been produced by thepancreas epithelium. Our data suggest that diagnosis based upon theproteome of pancreatic cyst fluid may include the expression of twohomologs of amylase, five mucins, five CEA-related cell adhesionmolecules (CEACAMs), and four S100 homologs. Furthermore, our studyindicates that proteomic profiling using small amounts of cyst fluidscan be a valuable tool for diagnosing and risk-stratifying cysticlesions of the pancreas.

Bodily fluids aspired from pancreatic cysts contain hundreds ofdifferent proteins. Some of the proteins are natural pancreatic enzymesecretions; others are plasma derived; yet others may be released by thecyst epithelium either normally or as a result of cellulartransformation. Proteomics by mass spectrometry provide a means toquickly quantify hundreds of these proteins simultaneously from a smallvolume of fluids. The identification of proteins that change theirlevels upon cellular transformation provides biomarkers for pancreasmalignancy.

We have determined that the risk of pancreatic cancer increases when theproteomic analysis shows an increase in several combinations ofbiomarkers: (1) When several isoforms of mucins 1, 2, 5AC, 5B, and 6 arepresent; (2) when mucins are present, the risk further increases whenisoforms of CEA, including CEACAM 1, 6, 7, and 8 are present; (3) whenmucins and CEA are either present or absent, the risk increases whenCEACAM8 is present and when certain of the biomarkers S100-A6, A8, A9,or A11 are present.

The biomarkers of the invention include genes and proteins, and variantsand fragments thereof. Such biomarkers include DNA comprising the entireor partial sequence of the nucleic acid sequence encoding the biomarker,or the complement of such a sequence. The biomarker nucleic acids alsoinclude RNA comprising the entire or partial sequence of any of thenucleic acid sequences of interest. A biomarker protein is a proteinencoded by or corresponding to a DNA biomarker of the invention. Abiomarker protein comprises the entire or partial amino acid sequence ofany of the biomarker proteins or polypeptides.

A “biomarker” is any gene or protein whose level of expression in atissue or cell is altered compared to that of a normal or healthy cellor tissue. Biomarkers of the invention are selective for underlying riskof progression to pancreatic cancer. By “selectively overexpressed inpancreatic cyst fluid” is intended that the biomarker of interest isoverexpressed in neoplastic cysts relative to benign or non-malignantcysts. Thus, detection of the biomarkers of the invention permits thedifferentiation of samples indicative of increased risk of developingneoplasms of the pancreas from samples that are indicative of benignproliferation. Representative biomarkers for pancreatic celltransformation include one or more or a plurality of the followingproteins:

Mucin-5B precursor—Homo sapiens (Human)Mucin-5AC—Homo sapiens (Human)Mucin-1 precursor—Homo sapiens (Human)Gelsolin precursor—Homo sapiens (Human)Carcinoembryonic antigen-related cell adhesion molecule 5 precursor—Homosapiens (Human)Ezrin—Homo sapiens (Human)Galectin-3-binding protein precursor—Homo sapiens (Human)Mucin-13 precursor—Homo sapiens (Human)Leukocyte elastase inhibitor—Homo sapiens (Human)Annexin A1—Homo sapiens (Human)Annexin A2—Homo sapiens (Human)Carcinoembryonic antigen-related cell adhesion molecule 6 precursor—Homosapiens (Human)Annexin A3—Homo sapiens (Human)Annexin A4—Homo sapiens (Human)Galectin-4—Homo sapiens (Human)Annexin A5—Homo sapiens (Human)Phosducin—Homo sapiens (Human)Tetraspanin-8—Homo sapiens (Human)Galectin-3—Homo sapiens (Human)Neutrophil gelatinase-associated lipocalin precursor—Homo sapiens(Human)Anterior gradient protein 2 homolog precursor—Homo sapiens (Human)Protein S100-A11—Homo sapiens (Human)Protein S100-A6—Homo sapiens (Human)Protein S100-A8—Homo sapiens (Human)Protein S100-A9—Homo sapiens (Human

Expression of the biomarkers described herein is indicative of cystfluid protein profiles that are associated with benign pancreaticdisease, pre-malignancy, and neoplastic lesions of the pancreas.

The phrase “genetic signature” refers to a plurality of nucleic acidmolecules whose expression levels are indicative of a given metabolic orpathological state. The genetic signatures described herein can beemployed to characterize at the molecular level the condition of thepancreatic cyst that is associated with an increased risk of pancreaticcancer, thus providing a useful molecular tool for predicting outcomes,for identifying patients at risk, and for use in biomarker in assays forevaluating cancer preventive agents.

For purposes of the present invention, “a” or “an” entity refers to oneor more of that entity; for example, “a cDNA” refers to one or more cDNAor at least one cDNA. The terms “a” or “an,” “one or more” and “at leastone” can be used interchangeably herein. It is also noted that the terms“comprising,” “including,” and “having” can be used interchangeably.Furthermore, a compound “selected from the group consisting of” refersto one or more of the compounds in the list that follows, includingmixtures (i.e. combinations) of two or more of the compounds. Accordingto the present invention, an isolated, or biologically pure molecule isa compound that has been removed from its natural milieu. As such,“isolated” and “biologically pure” do not necessarily reflect the extentto which the compound has been purified. An isolated compound of thepresent invention can be obtained from its natural source, can beproduced using laboratory synthetic techniques or can be produced by anysuch chemical synthetic route.

The term “genetic alteration” as used herein refers to a change from thewild-type or reference sequence of one or more nucleic acid molecules.Genetic alterations include without limitation, base pair substitutions,additions and deletions of at least one nucleotide from a nucleic acidmolecule of known sequence.

The term “solid matrix” as used herein refers to any format, such asbeads, microparticles, a microarray, the surface of a microtitrationwell or a test tube, a dipstick or a filter. The material of the matrixmay be polystyrene, cellulose, latex, nitrocellulose, nylon,polyacrylamide, dextran or agarose.

“Sample” or “patient sample” or “biological sample” generally refers toa sample which may be tested for a particular molecule, preferably agenetic signature specific marker molecule, such as a marker shown inthe tables provided below. Samples may include but are not limited tocells, cyst fluids, body fluids, including blood, serum, plasma, urine,saliva, tears, pleural fluid and the like.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID NO. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the functional and novelcharacteristics of the sequence.

With regard to nucleic acids used in the invention, the term “isolatednucleic acid” is sometimes employed. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryote or eukaryote. An “isolated nucleic acidmolecule” may also comprise a cDNA molecule. An isolated nucleic acidmolecule inserted into a vector is also sometimes referred to herein asa recombinant nucleic acid molecule.

With respect to RNA molecules, the term “isolated nucleic acid”primarily refers to an RNA molecule encoded by an isolated DNA moleculeas defined above. Alternatively, the term may refer to an RNA moleculethat has been sufficiently separated from RNA molecules with which itwould be associated in its natural state (i.e., in cells or tissues),such that it exists in a “substantially pure” form. By the use of theterm “enriched” in reference to nucleic acid it is meant that thespecific DNA or RNA sequence constitutes a significantly higher fraction(2-5 fold) of the total DNA or RNA present in the cells or solution ofinterest than in normal cells or in the cells from which the sequencewas taken. This could be caused by a person by preferential reduction inthe amount of other DNA or RNA present, or by a preferential increase inthe amount of the specific DNA or RNA sequence, or by a combination ofthe two. However, it should be noted that “enriched” does not imply thatthere are no other DNA or RNA sequences present, just that the relativeamount of the sequence of interest has been significantly increased.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term “purified” in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural level,this level should be at least 2-5 fold greater, e.g., in terms ofmg/ml). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones can be obtained directly from total DNA or from total RNA.The cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones yields an approximately 10⁻⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated. Thus, theterm “substantially pure” refers to a preparation comprising at least50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, etc.). More preferably, the preparation comprises atleast 75% by weight, and most preferably 90-99% by weight, the compoundof interest. Purity is measured by methods appropriate for the compoundof interest.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA or RNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. For example, specific hybridization can refer to a sequencewhich hybridizes to any specific marker gene or nucleic acid, but doesnot hybridize to other human nucleotides. Also polynucleotide which“specifically hybridizes” may hybridize only to a specific marker, sucha genetic signature-specific marker shown in the Tables below.Appropriate conditions enabling specific hybridization of singlestranded nucleic acid molecules of varying complementarity are wellknown in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al., Molecular Cloning, Cold Spring Harbor Laboratory (1989):

T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5°C. with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated T_(m) of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the T_(m) of the hybrid. In regards tothe nucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

The term “oligonucleotide” or “oligo” as used herein means a shortsequence of DNA or DNA derivatives typically 8 to 35 nucleotides inlength, primers, or probes. An oligonucleotide can be derivedsynthetically, by cloning or by amplification. An oligo is defined as anucleic acid molecule comprised of two or more ribo- ordeoxyribonucleotides, preferably more than three. The exact size of theoligonucleotide will depend on various factors and on the particularapplication and use of the oligonucleotide. The term “derivative” isintended to include any of the above described variants when comprisingan additional chemical moiety not normally a part of these molecules.These chemical moieties can have varying purposes including, improvingsolubility, absorption, biological half life, decreasing toxicity andeliminating or decreasing undesirable side effects.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be complementary to different strands of aparticular target nucleic acid sequence. This means that the probes mustbe sufficiently complementary so as to be able to “specificallyhybridize” or anneal with their respective target strands under a set ofpre-determined conditions. Therefore, the probe sequence need notreflect the exact complementary sequence of the target. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos.4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which areincorporated by reference herein.

An “siRNA” refers to a molecule involved in the RNA interference processfor a sequence-specific post-transcriptional gene silencing or geneknockdown by providing small interfering RNAs (siRNAs) that has homologywith the sequence of the targeted gene. Small interfering RNAs (siRNAs)can be synthesized in vitro or generated by ribonuclease III cleavagefrom longer dsRNA and are the mediators of sequence-specific mRNAdegradation. Preferably, the siRNA of the invention are chemicallysynthesized using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer. The siRNA canbe synthesized as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions. Commercial suppliersof synthetic RNA molecules or synthesis reagents include AppliedBiosystems (Foster City, Calif., USA), Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).Specific siRNA constructs for inhibiting elevated mRNA levels associatedwith pancreatic cancer may be between 15-35 nucleotides in length, andmore typically about 21 nucleotides in length.

The term “vector” relates to a single or double stranded circularnucleic acid molecule that can be infected, transfected or transformedinto cells and replicate independently or within the host cell genome. Acircular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that are targeted by restriction enzymes are readily availableto those skilled in the art, and include any replicon, such as aplasmid, cosmid, bacmid, phage or virus, to which another geneticsequence or element (either DNA or RNA) may be attached so as to bringabout the replication of the attached sequence or element. A nucleicacid molecule of the invention can be inserted into a vector by cuttingthe vector with restriction enzymes and ligating the two piecestogether.

Many techniques are available to those skilled in the art to facilitatetransformation, transfection, or transduction of the expressionconstruct into a prokaryotic or eukaryotic organism. The terms“transformation”, “transfection”, and “transduction” refer to methods ofinserting a nucleic acid and/or expression construct into a cell or hostorganism. These methods involve a variety of techniques, such astreating the cells with high concentrations of salt, an electric field,or detergent, to render the host cell outer membrane or wall permeableto nucleic acid molecules of interest, microinjection,peptide-tethering, PEG-fusion, and the like.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. In oneembodiment, the promoter element of the present invention precedes the5′ end of the pancreatic cancer specific marker nucleic acid molecule(s)such that the latter is transcribed into mRNA. Host cell machinery thentranslates mRNA into a polypeptide.

Those skilled in the art will recognize that a nucleic acid vector cancontain nucleic acid elements other than the promoter element and thepancreatic cancer specific marker gene nucleic acid molecule(s). Theseother nucleic acid elements include, but are not limited to, origins ofreplication, ribosomal binding sites, nucleic acid sequences encodingdrug resistance enzymes or amino acid metabolic enzymes, and nucleicacid sequences encoding secretion signals, localization signals, orsignals useful for polypeptide purification.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, plastid, phage or virus that is capable of replication largelyunder its own control. A replicon may be either RNA or DNA and may besingle or double stranded.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radio immunoassay, or bycolorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

The introduced nucleic acid may or may not be integrated (covalentlylinked) into nucleic acid of the recipient cell or organism. Inbacterial, yeast, plant and mammalian cells, for example, the introducednucleic acid may be maintained as an episomal element or independentreplicon such as a plasmid. Alternatively, the introduced nucleic acidmay become integrated into the nucleic acid of the recipient cell ororganism and be stably maintained in that cell or organism and furtherpassed on or inherited to progeny cells or organisms of the recipientcell or organism. Finally, the introduced nucleic acid may exist in therecipient cell or host organism only transiently.

The term “selectable marker gene” refers to a gene that when expressedconfers a selectable phenotype, such as antibiotic resistance, on atransformed cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of the coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and othertranscription control elements (e.g. enhancers) in an expression vector.

The terms “recombinant organism,” or “transgenic organism” refer toorganisms which have a new combination of genes or nucleic acidmolecules. A new combination of genes or nucleic acid molecules can beintroduced into an organism using a wide array of nucleic acidmanipulation techniques available to those skilled in the art. The term“organism” relates to any living being comprised of a least one cell. Anorganism can be as simple as one eukaryotic cell or as complex as amammal. Therefore, the phrase “a recombinant organism” encompasses arecombinant cell, as well as eukaryotic and prokaryotic organism.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated genetic signature nucleic acid or biomarkermolecule of the invention. Alternatively, this term may refer to aprotein that has been sufficiently separated from other proteins withwhich it would naturally be associated, so as to exist in “substantiallypure” form. “Isolated” is not meant to exclude artificial or syntheticmixtures with other compounds or materials, or the presence ofimpurities that do not interfere with the fundamental activity and thatmay be present, for example, due to incomplete purification, addition ofstabilizers, or compounding into, for example, immunogenic preparationsor pharmaceutically acceptable preparations.

A “specific binding pair” comprises a specific binding member (sbm) anda binding partner (bp) which have a particular specificity for eachother and which in normal conditions bind to each other in preference toother molecules. Examples of specific binding pairs are antigens andantibodies, ligands and receptors and complementary nucleotidesequences. The skilled person is aware of many other examples. Further,the term “specific binding pair” is also applicable where either or bothof the specific binding member and the binding partner comprise a partof a large molecule. In embodiments in which the specific binding paircomprises nucleic acid sequences, they will be of a length to hybridizeto each other under conditions of the assay, preferably greater than 10nucleotides long, more preferably greater than 15 or 20 nucleotideslong.

“Sample” or “patient sample” or “biological sample” generally refers toa sample which may be tested for a particular molecule or combination ofmolecules, preferably a combination of the biomarker or geneticsignature marker molecules, such as a combination of the markers shownin the Tables below. Samples may include but are not limited to cells,cyst fluids, body fluids, including blood, serum, plasma, urine, saliva,tears, pleural fluid and the like.

The terms “agent” and “test compound” are used interchangeably hereinand denote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materialssuch as bacteria, plants, fungi, or animal (particularly mammalian)cells or tissues. Biological macromolecules include siRNA, shRNA,antisense oligonucleotides, small molecules, antibodies, peptides,peptide/DNA complexes, and any nucleic acid based molecule, for examplean oligo, which exhibits the capacity to modulate the activity of thegenetic signature nucleic acids described herein or their encodedproteins. Agents are evaluated for potential biological activity byinclusion in screening assays described herein below.

The term “modulate” as used herein refers increasing or decreasing. Forexample, the term modulate refers to the ability of a compound or testagent to either interfere with, or augment signaling or activity of agene or protein of the present invention.

Methods of Using the Biomarkers and Genetic Signatures of the Invention

Genetic signature or biomarker encoding nucleic acids, including but notlimited to those listed in the Tables hereinbelow may be used for avariety of purposes in accordance with the present invention. Thegenetic signature associated with an increased risk of pancreatic cancer(e.g., the plurality of nucleic acids contained therein) containing DNA,RNA, or fragments thereof may be used as probes to detect the presenceof and/or expression of these specific markers in a biological sample.Methods in which such marker nucleic acids may be utilized as probes forsuch assays include, but are not limited to: (1) in situ hybridization;(2) Southern hybridization (3) northern hybridization; and (4) assortedamplification reactions such as polymerase chain reactions (PCR).

Further, assays for detecting the genetic signature may be conducted onany type of biological sample, but is most preferably performed on cystfluid. From the foregoing discussion, it can be seen that geneticsignature containing nucleic acids, vectors expressing the same, geneticsignature encoded proteins and anti-genetic signature encoded proteinspecific antibodies of the invention can be used to detect the signaturein body tissue, cells, or fluid, and alter genetic signature containingmarker protein expression for purposes of assessing the genetic andprotein interactions involved in pancreatic cancer.

In certain embodiments for screening for genetic signature containingnucleic acid(s), the sample will initially be amplified, e.g. using PCR,to increase the amount of the template as compared to other sequencespresent in the sample. This allows the target sequences to be detectedwith a high degree of sensitivity if they are present in the sample.This initial step may be avoided by using highly sensitive arraytechniques that are becoming increasingly important in the art.

Alternatively, alternative detection technologies will be employed whichdetect the pancreatic cancer biomarker proteins directly. Such methodsinclude geLC/MS/MS proteomics analysis. This approach provides a fullpanel of the protein biomarkers present in cyst fluid and allows theclinician to predict outcomes based on the panel of biomarkers presentin a sample.

Thus, any of the aforementioned techniques may be used to detect orquantify genetic signature expression and or protein expression levelsand accordingly, diagnose patient susceptibility for developingpancreatic cancer.

Kits and Articles of Manufacture

Any of the aforementioned products can be incorporated into a kit whichmay contain genetic signature polynucleotides or one or more suchmarkers immobilized on a Gene Chip, an oligonucleotide, a polypeptide, apeptide, an antibody, a label, marker, or reporter, a pharmaceuticallyacceptable carrier, a physiologically acceptable carrier, instructionsfor use, a container, a vessel for administration, an assay substrate,or any combination thereof.

Methods of Using the Genetic Signature or Biomarker Proteins forDevelopment of Therapeutic Agents

Since the genetic signature identified herein and the proteins encodedthereby has been associated with the etiology of pancreatic cancer,methods for identifying agents that modulate the activity of the genesand their encoded products should result in the generation ofefficacious therapeutic agents for the treatment of a cancer,particularly pancreatic cancer.

The nucleic acids comprising the signature contain regions which providesuitable targets for the rational design of therapeutic agents whichmodulate their activity. Small peptide molecules corresponding to theseregions may be used to advantage in the design of therapeutic agentswhich effectively modulate the activity of the encoded proteins.Molecular modeling should facilitate the identification of specificorganic molecules with capacity to bind to the active site of theproteins encoded by the genetic signature nucleic acids based onconformation or key amino acid residues required for function. Acombinatorial chemistry approach will be used to identify molecules withgreatest activity and then iterations of these molecules will bedeveloped for further cycles of screening. In certain embodiments,candidate agents can be screening from large libraries of synthetic ornatural compounds. Such compound libraries are commercially availablefrom a number of companies including but not limited to MaybridgeChemical Co., (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.),Microsour (New Milford, Conn.) Aldrich (Milwaukee, Wis.) Akos Consultingand Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France),Asinex (Moscow, Russia) Aurora (Graz, Austria), BioFocus DPI(Switzerland), Bionet (Camelford, UK), Chembridge (San Diego, Calif.),Chem Div (San Diego, Calif.). The skilled person is aware of othersources and can readily purchase the same. Once therapeuticallyefficacious compounds are identified in the screening assays describedherein, they can be formulated in to pharmaceutical compositions andutilized for the treatment of pancreatic cancer.

The polypeptides or fragments employed in drug screening assays mayeither be free in solution, affixed to a solid support or within a cell.One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant polynucleotidesexpressing the biomarker polypeptide or fragment, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may determine, for example,formation of complexes between the polypeptide or fragment and the agentbeing tested, or examine the degree to which the formation of a complexbetween the polypeptide or fragment and a known substrate is interferedwith by the agent being tested.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity for the encodedpolypeptides and is described in detail in Geysen, PCT publishedapplication WO 84/03564, published on Sep. 13, 1984. Briefly stated,large numbers of different, small peptide test compounds, such as thosedescribed above, are synthesized on a solid substrate, such as plasticpins or some other surface. The peptide test compounds are reacted withthe target polypeptide and washed. Bound polypeptide is then detected bymethods well known in the art.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have anonfunctional or altered pancreatic cancer associated gene. These hostcell lines or cells are defective at the polypeptide level. The hostcell lines or cells are grown in the presence of drug compound. Theeffect on cellular morphology and/or proliferation of the host cells ismeasured to determine if the compound is capable of regulating the samein the defective cells. Host cells contemplated for use in the presentinvention include but are not limited to bacterial cells, fungal cells,insect cells, mammalian cells, particularly pancreatic cells. Thegenetic signature encoding DNA molecules may be introduced singly intosuch host cells or in combination to assess the phenotype of cellsconferred by such expression. Methods for introducing DNA molecules arealso well known to those of ordinary skill in the art. Such methods areset forth in Ausubel et al. eds., Current Protocols in MolecularBiology, John Wiley & Sons, NY, N.Y. 1995, the disclosure of which isincorporated by reference herein.

Pancreatic cells and pancreatic cell lines suitable for studying theeffects of genetic signature expression on cellular morphology andsignaling methods of use thereof for drug discovery are provided. Suchcells and cell lines will be transfected with genetic signature encodingnucleic acids described herein and the effects on pancreatic cellfunctions and/or cyst formation can be determined. Such cells and celllines can also be contacted with the siRNA molecules provided herein toassess the effects thereof on malignant transformation. The siRNAmolecules will be tested alone and in combination of 2, 3, 4, and 5siRNAs to identify the most efficacious combination for down regulatingtarget nucleic acids.

A wide variety of expression vectors are available that can be modifiedto express the novel DNA or RNA sequences of this invention. Thespecific vectors exemplified herein are merely illustrative, and are notintended to limit the scope of the invention. Expression methods aredescribed by Sambrook et al. Molecular Cloning: A Laboratory Manual orCurrent Protocols in Molecular Biology 16.3-17.44 (1989). Expressionmethods in Saccharomyces are also described in Current Protocols inMolecular Biology (1989).

Suitable vectors for use in practicing the invention include prokaryoticvectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey PinesRd., La Jolla, Calif. 92037), pET vectors (Novogen Inc., 565 ScienceDr., Madison, Wis. 53711) and the pGEX vectors (Pharmacia LKBBiotechnology Inc., Piscataway, N.J. 08854). Examples of eukaryoticvectors useful in practicing the present invention include the vectorspRc/CMV, pRc/RSV, and pREP (Invitrogen, 11588 Sorrento Valley Rd., SanDiego, Calif. 92121); pcDNA3.11V5&His (Invitrogen); baculovirus vectorssuch as pVL1392, pVL1393, or pAC360 (Invitrogen); and yeast vectors suchas YRP17, YIP5, and YEP24 (New England Biolabs, Beverly, Mass.), as wellas pRS403 and pRS413 Stratagene Inc.); Picchia vectors such as pHIL-D1(Phillips Petroleum Co., Bartlesville, Okla. 74004); retroviral vectorssuch as PLNCX and pLPCX (Clontech); and adenoviral and adeno-associatedviral vectors.

Promoters for use in expression vectors of this invention includepromoters that are operable in prokaryotic or eukaryotic cells.Promoters that are operable in prokaryotic cells include lactose (lac)control elements, bacteriophage lambda (pL) control elements, arabinosecontrol elements, tryptophan (trp) control elements, bacteriophage T7control elements, and hybrids thereof. Promoters that are operable ineukaryotic cells include Epstein Barr virus promoters, adenoviruspromoters, SV40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus(CMV) promoters, baculovirus promoters such as AcMNPV polyhedrinpromoter, Picchia promoters such as the alcohol oxidase promoter, andSaccharomyces promoters such as the gal4 inducible promoter and the PGKconstitutive promoter, as well as neuronal-specific platelet-derivedgrowth factor promoter (PDGF).

In addition, a vector of this invention may contain any one of a numberof various markers facilitating the selection of a transformed hostcell. Such markers include genes associated with temperaturesensitivity, drug resistance, or enzymes associated with phenotypiccharacteristics of the host organisms.

Host cells expressing the genetic signature of the present invention orfunctional fragments thereof provide a system in which to screenpotential compounds or agents for the ability to modulate thedevelopment of pancreatic cancer

Another approach entails the use of phage display libraries engineeredto express fragment of the polypeptides encoded by the genetic signaturecontaining nucleic acids on the phage surface. Such libraries are thencontacted with a combinatorial chemical library under conditions whereinbinding affinity between the expressed peptide and the components of thechemical library may be detected. U.S. Pat. Nos. 6,057,098 and 5,965,456provide methods and apparatus for performing such assays.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology9:19-21. In one approach, discussed above, the three-dimensionalstructure of a protein of interest or, for example, of theprotein-substrate complex, is solved by x-ray crystallography, bynuclear magnetic resonance, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson et al., (1990)Science 249:527-533). In addition, peptides may be analyzed by analanine scan (Wells, (1991) Meth. Enzym. 202:390-411). In thistechnique, an amino acid residue is replaced by Ala, and its effect onthe peptide's activity is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner to determine the importantregions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacophore upon which subsequent drug designcan be based.

One can bypass protein crystallography altogether by generatinganti-idiotypic antibodies (anti-ids) to a functional, pharmacologicallyactive antibody. As a mirror image of a mirror image, the binding siteof the anti-ids would be expected to be an analog of the originalmolecule. The anti-id could then be used to identify and isolatepeptides from banks of chemically or biologically produced banks ofpeptides. Selected peptides would then act as the pharmacophore.

Thus, one may design drugs which have, e.g., improved polypeptideactivity or stability or which act as inhibitors, agonists, antagonists,etc. of polypeptide activity. By virtue of the availability of thegenetic signature containing nucleic acid sequences described herein,sufficient amounts of the encoded polypeptide may be made available toperform such analytical studies as x-ray crystallography. In addition,the knowledge of the protein sequence provided herein will guide thoseemploying computer modeling techniques in place of, or in addition tox-ray crystallography.

In another embodiment, the availability of genetic signature containingnucleic acids enables the production of strains of laboratory micecarrying the signature(s) of the invention. Transgenic mice expressingthe genetic signature of the invention provide a model system in whichto examine the role of the protein(s) encoded by the signaturecontaining nucleic acid in the development and progression towardspancreatic cancer. Methods of introducing transgenes in laboratory miceare known to those of skill in the art. Three common methods include:(1) integration of retroviral vectors encoding the foreign gene ofinterest into an early embryo; (2) injection of DNA into the pronucleusof a newly fertilized egg; and (3) the incorporation of geneticallymanipulated embryonic stem cells into an early embryo. Production of thetransgenic mice described above will facilitate the molecularelucidation of the role that a target protein plays in various cellularmetabolic processes. Such mice provide an in vivo screening tool tostudy putative therapeutic drugs in a whole animal model and areencompassed by the present invention.

The term “animal” is used herein to include all vertebrate animals,except humans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. A “transgenic animal”is any animal containing one or more cells bearing genetic informationaltered or received, directly or indirectly, by deliberate geneticmanipulation at the subcellular level, such as by targeted recombinationor microinjection or infection with recombinant virus. The term“transgenic animal” is not meant to encompass classical cross-breedingor in vitro fertilization, but rather is meant to encompass animals inwhich one or more cells are altered by or receive a recombinant DNAmolecule. This molecule may be specifically targeted to a definedgenetic locus, be randomly integrated within a chromosome, or it may beextra-chromosomally replicating DNA. The term “germ cell line transgenicanimal” refers to a transgenic animal in which the genetic alteration orgenetic information was introduced into a germ line cell, therebyconferring the ability to transfer the genetic information to offspring.If such offspring, in fact, possess some or all of that alteration orgenetic information, then they, too, are transgenic animals.

The alteration of genetic information may be foreign to the species ofanimal to which the recipient belongs, or foreign only to the particularindividual recipient, or may be genetic information already possessed bythe recipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene. Such altered or foreigngenetic information would encompass the introduction of geneticsignature containing nucleotide sequences.

The DNA used for altering a target gene may be obtained by a widevariety of techniques that include, but are not limited to, isolationfrom genomic sources, preparation of cDNAs from isolated mRNA templates,direct synthesis, or a combination thereof.

A preferred type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells may be obtained from pre-implantationembryos cultured in vitro (Evans et al., (1981) Nature 292:154-156;Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc.Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introducedinto the ES cells by standard techniques such as DNA transfection or byretrovirus-mediated transduction. The resultant transformed ES cells canthereafter be combined with blastocysts from a non-human animal. Theintroduced ES cells thereafter colonize the embryo and contribute to thegerm line of the resulting chimeric animal.

One approach to the problem of determining the contributions ofindividual genes and their expression products is to use geneticsignature associated genes as insertional cassettes to selectivelyinactivate a wild-type gene in totipotent ES cells (such as thosedescribed above) and then generate transgenic mice. The use ofgene-targeted ES cells in the generation of gene-targeted transgenicmice was described, and is reviewed elsewhere (Frohman et al., (1989)Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).

Techniques are available to inactivate or alter any genetic region to amutation desired by using targeted homologous recombination to insertspecific changes into chromosomal alleles. However, in comparison withhomologous extra-chromosomal recombination, which occurs at a frequencyapproaching 100%, homologous plasmid-chromosome recombination wasoriginally reported to only be detected at frequencies between 10⁻⁶ and10⁻³. Non-homologous plasmid-chromosome interactions are more frequentoccurring at levels 10⁵-fold to 10² fold greater than comparablehomologous insertion.

To overcome this low proportion of targeted recombination in murine EScells, various strategies have been developed to detect or rarehomologous recombinants. One approach for detecting homologousalteration events uses the polymerase chain reaction (PCR) to screenpools of transformant cells for homologous insertion, followed byscreening of individual clones. Alternatively, a positive geneticselection approach has been developed in which a marker gene isconstructed which will only be active if homologous insertion occurs,allowing these recombinants to be selected directly. One of the mostpowerful approaches developed for selecting homologous recombinants isthe positive-negative selection (PNS) method developed for genes forwhich no direct selection of the alteration exists. The PNS method ismore efficient for targeting genes which are not expressed at highlevels because the marker gene has its own promoter. Non-homologousrecombinants are selected against by using the Herpes Simplex virusthymidine kinase (HSV-TK) gene and selecting against its nonhomologousinsertion with effective herpes drugs such as gancyclovir (GANC) or(1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodou-racil, (FIAU). Bythis counter selection, the number of homologous recombinants in thesurviving transformants can be increased. Utilizing genetic signaturecontaining nucleic acid as a targeted insertional cassette providesmeans to detect a successful insertion as visualized, for example, byacquisition of immunoreactivity to an antibody immunologically specificfor the polypeptide encoded genetic signature nucleic acid(s) and,therefore, facilitates screening/selection of ES cells with the desiredgenotype.

As used herein, a knock-in animal is one in which the endogenous murinegene, for example, has been replaced with human geneticsignature-associated gene(s) of the invention. Such knock-in animalsprovide an ideal model system for studying the development of pancreaticcancer.

As used herein, the expression of a genetic signature containing nucleicacid, fragment thereof, or genetic signature fusion protein can betargeted in a “tissue specific manner” or “cell type specific manner”using a vector in which nucleic acid sequences encoding all or a portionof genetic signature-associated protein are operably linked toregulatory sequences (e.g., promoters and/or enhancers) that directexpression of the encoded protein in a particular tissue or cell type.Such regulatory elements may be used to advantage for both in vitro andin vivo applications. Promoters for directing tissue specific expressionof proteins are well known in the art and described herein.

Methods of use for the transgenic mice of the invention are alsoprovided herein. Transgenic mice into which a nucleic acid containingthe genetic signature or its encoded protein(s) have been introduced areuseful, for example, to develop screening methods to screen therapeuticagents to identify those capable of modulating the development ofpancreatic cancer.

Pharmaceuticals and Peptide Therapies

The elucidation of the role played by the gene products described hereinin pancreatic cancer progression facilitates the development ofpharmaceutical compositions useful for treatment and diagnosis ofpancreatic cancer. These compositions may comprise, in addition to oneof the above substances, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,small molecule or other pharmaceutically useful compound according tothe present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual.

As it is presently understood, RNA interference involves a multi-stepprocess. Double stranded RNAs are cleaved by the endonuclease Dicer togenerate nucleotide fragments (siRNA). The siRNA duplex is resolved into2 single stranded RNAs, one strand being incorporated into aprotein-containing complex where it functions as guide RNA to directcleavage of the target RNA (Schwarz et al, Mol. Cell. 10:537 548 (2002),Zamore et al, Cell 101:25 33 (2000)), thus silencing a specific geneticmessage (see also Zeng et al, Proc. Natl. Acad. Sci. 100:9779 (2003)).

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in parenteral, oral solid andliquid formulations, ophthalmic, suppository, aerosol, topical or othersimilar formulations. These pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Thus such compositions mayoptionally contain other components, such as adjuvants, e.g., aqueoussuspensions of aluminum and magnesium hydroxides, and/or otherpharmaceutically acceptable carriers, such as saline. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to administer theappropriate agent to a patient according to the methods of theinvention. The use of nanoparticles to deliver agents, as well as cellmembrane permeable peptide carriers that can be used are described inCrombez et al., Biochemical Society Transactions v 35:p 44 (2007).

In order to treat an individual having pancreatic cancer, to alleviate asign or symptom of the disease, the pharmaceutical agents of theinvention should be administered in an effective dose. The totaltreatment dose can be administered to a subject as a single dose or canbe administered using a fractionated treatment protocol, in whichmultiple doses are administered over a more prolonged period of time,for example, over the period of a day to allow administration of a dailydosage or over a longer period of time to administer a dose over adesired period of time. One skilled in the art would know that theamount of agent required to obtain an effective dose in a subjectdepends on many factors, including the age, weight and general health ofthe subject, as well as the route of administration and the number oftreatments to be administered. In view of these factors, the skilledartisan would adjust the particular dose so as to obtain an effectivedose for treating an individual having pancreatic cancer.

In an individual suffering from pancreatic cancer, in particular a moresevere form of the disease, administration of agent can be particularlyuseful when administered in combination, for example, with aconventional agent for treating such a disease. The skilled artisanwould administer the agent alone or in combination and would monitor theeffectiveness of such treatment using routine methods such as sonogram,radiologic, immunologic or, where indicated, histopathologic methods.Other conventional agents for the treatment of pancreatic cancer includeanti cancer agents, such as gemcitabine and erlotinib. Administration ofthe pharmaceutical preparation is preferably in an “effective amount”this being sufficient to show benefit to the individual. This amountprevents, alleviates, abates, or otherwise reduces the severity ofpancreatic cancer symptoms in a patient.

The pharmaceutical preparation is formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of active ingredient calculated toproduce the desired effect in association with the selectedpharmaceutical carrier. Procedures for determining the appropriatedosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

The Examples below are provided to illustrate certain embodiments of theinvention. They are not intended to limit the invention in any way.

Example I Proteomic Analysis of Pancreatic Cancer Fluids

The following materials and methods are provided to facilitate thepractice of the present invention.

Sample Acquisition. Aliquots of cyst fluid that were used for thisproject were obtained from materials that were aspirated for clinicalpurposes. The study was approved by the Institutional Review Board ofthe Fox Chase Cancer Center. EUS-FNA (14) was performed under conscioussedation using a linear echoendoscope. When a lesion was identified,EUS-FNA was performed with a 22 or 19-gauge needle through either atransduodenal or transgastric approach, depending on the location of thelesion within the pancreas. The highest priority was given to procuringa volume of fluid that was adequate to perform the necessary clinicallyindicated diagnostic assays (e.g., cytology, CEA in ng/mL, Mayo MedicalLaboratories, code #84074), amylase (in units/L, Mayo MedicalLaboratories, code #5079). As little as 40 μL of cyst fluids per patientwere allocated for the proteomic study. For the purpose of this study,cyst fluid cytology findings were grouped into the following categories:A—Benign: No evidence of benign mucinous epithelium, atypical cells orcarcinoma; B—Benign mucinous epithelium; C—Atypical/suspicious cytology;D—Malignant.

Proteomics Analysis. Standard Operating Procedures were established andfollowed for all steps of cyst fluid collection and analysis. The cystfluid was diluted with three volumes of PBS, mixed, and centrifuged for10 minutes at 13,000×g at 4° C. to remove cells and any insolublematerials, snap frozen in liquid nitrogen in aliquots and banked at −80°C. To remove small peptides bound to larger proteins, the cyst fluid wastreated with three volumes of 0.1 M glycine pH 2.3 and acetonitrile wasadded to 25% v/v final concentration. The solution was filtered byultrafiltration (pre-washed Amicon YM-30 Centricon #4208) at 4000×g at4° C. for about one hour to reach minimum retention volume designed forthe unit. The retained proteins above the filter were solubilized with200 μL of 0.2% SDS solution and transferred to a 1.5 mL microcentrifugetube. Three volumes of cold acetone were added to precipitate theproteins overnight at −20° C. and the suspension was then centrifuged at21,000×g for 40 min. The pellet was washed once with 80% cold acetone,centrifuged, and air dried, then resolubilized in 2D PAGE sample buffer(7 M urea, 2 M thiourea, 4% (w/v) CHAPS). Protein concentration wasdetermined as previously described (15).

Protein (15 μg) was reduced with dithiothreitol and alkylated byiodoacetamide at 25° C. for 1 hr (15) and then resolved in a pre-castNovex 4-12% gradient PAGE with 3 mm wide wells (Invitrogen™, CA, USA).Electrophoresis was performed in MOPS buffer at 150V at room temperaturefor about 20 min until the tracking dye was 1.5 cm from the top of thegel. The gel cassette was opened in a laminar flow hood. Each samplelane, two per gel, was cut into 11 slices from the well to about 2 mmbeyond the dye front. Each gel slice was again subjected to reductionand alkylation. Porcine trypsin (Sigma proteomic grade #T6567) was addedas 63 ng in 7 μL 25 mM ammonium bicarbonate and incubated for 30 min.Unabsorbed trypsin of about 2 μL was removed and 20 μL of 25 mM ammoniumbicarbonate was added and incubated at 37° C. for about 16 hours. 10 μLof the peptide solution was mixed with 2.5 μL of 25% acetonitrile 1%formic acid, and 2 μL was injected into the LC/MS/MS system for proteinidentification. A LC/MS/MS system consisted of an Applied BiosystemsQSTAR XL hybrid quadruple TOF mass spectrometer supported by an AgilentnanoLC system. For 15 μg gel loading, 10% of the digest of each gelslice was auto-injected onto a trap column (Agilent Zorbax 300SB-C18, 5μm, 5×0.3 mm), washed, and eluted at 0.3 μL/min through an analyticalcolumn (Agilent Zorbax 300SB-C18, 3.5 μm, 150×0.1 mm) at roomtemperature. The elution gradient was in 0.2% formic acid with linearsegments of 4.5%, 4.5%, 28%, 54%, 90% acetonitrile at 0, 4, 8, 80, 85min, respectively. An IDA protocol using MS periods of 2 s of TOF-MS andthree cycles of 4 s of MS/MS each was used to obtain highly accuratespectra for protein identification for the three most intense peptideions in each cycle. For discovery of more proteins and peptides in cystfluids and to overcome the possibility of false-negatives due tounder-sampling of co-eluting peptides, an exclusion list of the peptidesin the first LC/MS/MS run of a cyst fluid was used to direct the secondLC/MS/MS run to sequence new peptides. The two peak lists were combinedfor database searching for protein identification and for relativequantitation of the proteins by emPAI score (exponentially modifiedprotein abundance index) without isotope labeling (16). The emPAI score,[10̂(# observed peptides/# theoretical peptides)−1], is roughlyproportional to the abundance of a protein in a complex mixture. Almostevery protein identified in Tables 1-3 was abundant enough to beidentified, and its relative abundance quantified for comparison, in thefirst LC/MS/MS run.

Results

Pancreatic cyst fluids were obtained by EUS from 20 patients forgeLC/MS/MS proteomics analysis. The proteins of pancreatic cyst fluidscan be subjected to proteolysis in some situations if the pancreaticproteases are inadvertently activated, and if inhibition by serumprotease inhibitors is ineffective. To avoid this problem, proteins thatwere larger than 10,000 in molecular weight were analyzed andquantification performed at the level of tryptic peptides. For theanalysis described herein, pancreatic cyst fluids appeared robust andstable, providing the same mass spectrometry information after multiplefreeze-thaw cycles. The samples were unaffected by room temperatureincubation (data not shown). However, cyst fluids are rich in smallpeptides bound to other carrier proteins in the sample. Ourfractionation procedure removed these small peptides (data not shown),simplified the mass spectra obtained by geLC/MS/MS, and significantlyincreased the sensitivity of biomarker detection.

Clinical Information on the Cyst Fluids. Demography of the patients,dimensions of the cysts, and the results of traditional clinical testsperformed on the cyst fluids are shown in Table 1. In Tables 1 to 4,because CEA measurements by clinical immunoassays are believed to be thestrongest indicators of mucinous versus non-mucinous cysts in theabsence of direct measurement of the mucins, the cysts are presented inthe order of increasing CEA. Two samples without CEA values were locatedon the right side because high CEA values would be anticipated based onthe histopathology findings.

All the patients were Caucasians. The various diagnostic assays,commercial amylase and CEA levels, were not obtained for all studypatients. These absent values are represented by empty boxes in Table 1.For cysts 17, 14, 20, 5, and 21, subsequent surgical resection led todefinitive histopathologic diagnosis as shown. Cyst 19B, diagnosed byhistology after surgical resection as an IPMN adenoma, was the samepatient as cyst 19A except the latter occurred five months before thesurgical resection, providing a view of the biomarker transition.

The pancreatic cyst fluid proteome. Samples were purified and analyzedby geLC/MS/MS as described in Materials and Methods. The cyst fluids inthis study vary in the amounts of plasma proteins versus pancreaticenzymes. About 137 proteins normally found in plasma were observed among13 of the pancreatic cyst fluids. A partial list of these proteins isshown in Table 2. Hemoglobin, IgG, serum albumin, apolipoprotein A1 andAII, and serotransferrin were among the most abundant serum proteinswhen present. Hemoglobin was found in significant quantities only infive of the 20 cysts, suggesting that there was minimal contamination ofblood from needle puncture during EUS-FNA collection of the cyst fluids.If red blood cells were present, they were successfully removed bycentrifugation. Eight of the cyst fluids that contained the most plasmainfiltration did not contain pancreatic enzymes. For example cysts 15and 1 contained only plasma proteins (Table 2), no detectable pancreaticenzymes (Table 3), no mucins, no CEACAM, and no S100 homologs (Table 4).Seven of the cyst fluid samples were essentially free of proteins fromblood. Most of these contained abundant pancreatic enzymes. Thedistribution of some of the 29 pancreatic enzymes among the cysts inthis study is shown in Table 3. These enzymes included digestive enzymesand proteins important to pancreatic function. The latter included thepancreatic stone protein Lithostathine 1, the Regenerating islet-derivedprotein 3 alpha that has multiple functions, and Pancreatic secretorygranule membrane major glycoprotein GP2. Amylase is not always observedin cysts that contained abundant levels of other pancreatic enzymes.

Data Analysis. Samples were purified and analyzed by geLC/MS/MS asdescribed above. The mass spectrometry “wiff” data files were used tosearch the SwissProt protein database release 54.1 using MASCOT 2.2(Matrix Sciences, London, U.K.), analyzing the MS/MS sequencing spectraof the +2 and +3 ions. Fixed modification of carbamidomethylcysteine,variable oxidation of methionine, and one trypsin miss were allowed forprotein identification, but the latter two were disallowed forcalculating the emPAI scores. Peptide mass tolerance was +/−150 ppm andfragment mass tolerance was 0.5 Da. False discovery rate was less than3.5% for individual peptides as judged by hits at a decoy database withrandomized sequences in each entry. Thus the confidence of correctprotein identification is very high when three or more unique peptideswith high quality sequencing spectra, and from the same position in thegel, are congruent in their identification of a protein in this project.

The presence of major protein classes of blood proteins, pancreaticenzymes, and keratins, in each sample, and the limited number ofdefinitively histopathologically identified samples in this study,confound effective classification of the potential biomarkers by typicalstatistical approaches that include unsupervised hierarchical clustering(17) and principal component analysis (18). Low abundance proteins withan emPAI score average for the expressing samples of less than 0.01 werefirst removed, leaving 466 proteins identified with confidence. ThisemPAI score represents about one peptide sequence identified in aprotein of about 250,000 molecular weight thus some of the lower scoreprotein identifications were within the approximately 3% false positiveidentification rate for this data. Next, 34 keratins, 137 bloodproteins, and 29 pancreatic enzymes, were filtered from the proteome ofeach cyst fluid sample. The remaining 295 proteins were sorted by theaverage emPAI score calculated from the samples expressing each protein.Among the most abundant ones in this list of pancreatic cyst fluidproteins were the homologs of three families of proteins previouslyproposed to be biomarkers of pancreatic cancer, namely mucins, CEACAM's(19), and S100's (20-22).

Proteomics Biomarkers. Several biomarkers, some of whose homologs areknown to be elevated in pancreatic cancer, were identified in the cystfluids (Table 4). Ten of the cyst fluids contained one or more mucinhomologs, some of which have low amino acid sequence homology to eachother. Cyst fluid from seven of the patients revealed the presence ofCEACAM homologs by mass spectrometry detection. Five of the patientsshowed expression of S100 protein homologs in their cyst fluid. Therelative abundances of CEACAM5 (CEA) determined by emPAI score were inrough agreement with the clinical assays performed on the samples shownin Table 1, bearing in mind the differences in CEA measurementprocedures. More specifically, the emPAI score for CEACAM5 wasdetermined as score per unit protein used in mass spectrometry while theclinical immunoassay CEA unit was concentration in ng per mL cyst fluid.In each case where the identification of a proteomic biomarker was atlow abundance in a given cyst fluid, we ruled out the possibility ofsample carry over by verifying that the same biomarker was not detectedin the cyst fluid loaded onto the HPLC column in the preceding sample.

Discussion

Pancreatic cyst fluid aspired via EUS-FNA are used clinically to providebiomarkers that facilitate the diagnosis of the potential of pancreaticcancer in patients. The number of assays feasible for each patient isoften limited by the quantity of cyst fluids available which is partly afunction of the cyst size. For example, the volume of cyst fluidsrequired to submit to either the clinical amylase assay or the clinicalCEA assay used in this study is 0.5 mL. Moreover, cytologic diagnosis isfacilitated using as large a cyst fluid sample as possible. The scarcityof cyst fluids in cysts smaller than one centimeter in diameter is oneof the reasons why such small cysts are often not referred to EUS forevaluation. Thus, a new assay that provides for the measurement ofmucins, amylase, and CEA in a minute volume of fluid, providesclinically relevant information in situations where the cyst fluidvolumes are small. The proteome of pancreatic cyst fluids as elucidatedby LC/MS/MS mass spectrometry proteomics provides comprehensiveinformation on cancer biomarkers in pancreatic cyst fluids using aminimal volume of fluids. Interesting observations made on four classesof biomarkers are described below.

Amylase biomarker. Although the measurement of amylase in blood has beena traditional biomarker of pancreatitis, the basis for using amylasemeasurements in pancreatic cyst fluid as an indicator of pancreatitis,non-mucinous cyst, or the absence of cancer has not been well studied.We show here that the pancreatic amylase activity in the cyst fluids isdivided into two isozymes, alpha amylase 2B and pancreatic alphaamylase, encoded by two separate genes, AMY2B and AMY2A, respectively.The two isozymes have 98% sequence identity, but may differ in theirregulation as shown in cysts 8, 10, 19B, and 14. Thus it may bepertinent to consider the levels of the two amylase isozymesindividually. Amylase by itself is not always a good indicator of thepresence of pancreatic enzymes as in the cases of 19B, cyst 3 and cyst14 (Table 3). Although pancreatic lipases have been suggested as asubstitute for amylase in the analysis of pancreatic cyst fluids,carboxypeptidases A1 and B may be equally effective as indicators of thepresence of pancreatic enzymes in this set of samples. The simultaneousmeasurement of many pancreatic enzymes, made feasible by the use of massspectrometry proteomics, may provide more complete information withoutthe limitation of choosing one pancreatic enzyme as biomarker.

Abnormal expression of mucins and changes in their post-translationalmodification patterns have long been recognized as potential biomarkersof malignancy (3, 23). Table 4 shows that five soluble mucin homologs incysts 2, 11, 9, and 19 can be distinguished and conveniently measuredvia LC/MS/MS proteomics and may assist in future classification ofcysts. Soluble mucins were detected in these cases where the cytologistswere unable to detect mucinous epithelial cells.

CEACAM biomarkers. There are at least seven carcinoembryonic antigenhomologs in humans (24, 25). The widely used CEA in clinical tests forvarious cancers (26) and in pancreatic cyst fluids is CEACAM5 (Table 1).For example, CEA levels of >400 ng/mL appear to be specific formucin-producing cystic neoplasms (27). However, the CEA levels in thesetumors is frequently lower, thus using a cutoff of 400 ng/mL may resultin an unacceptably high “miss rate” for diagnosing these potentiallymalignant tumors (27). Alternatively, a level of 192 ng/mL has beencited as the “optimal” cutoff value (i.e., provides the best combinedsensitivity and specificity for distinguishing mucinous fromnon-mucinous pancreatic cysts); however, this value results in anaccuracy rate of only 79% (3). For example, cyst 14 had a CEA level of582 ng/mL and amylase of 2853 U/L via clinical assays (Table 1) wasfound to be a MCA upon surgical histopathology. Accordingly, proteomicsshowed that there was down-regulation of amylase in this enzyme cyst(Table 2) plus higher levels of the proteins of CEA homolog CEACAM6 andCEACAM7 than CEA (Table 4). Thus it appears that a combined high levelof the CEACAM homologs are as important an indication as a high level ofCEA by itself. As discussed below, the expression of S100A6 and S100A9in this cyst are indicative of further progression for this neoplasm(Table 4). Cyst 3 with a clinical CEA assay of 63,830 had declined aWhipple procedure and thus had no pathology information. This high CEAvalue is consistent with its high mucin content and high CEACAM 5 andCEACAM 6 seen in proteomics (Table 4). However, the absence of S100expression distinguish it from cysts 5, 14, and 21. Thus CEACAM homologsare markers that can assist in risk-stratifying pancreatic cysts.

S100 biomarkers. The S100 protein family includes small Ca⁺⁺ bindingproteins that are soluble in 100% saturated ammonium sulfate solutionand have long been recognized as biomarkers of brain cancer. A recentreview provides references to the many cellular functions in which S100homologs appear to participate (20). Although S100S8, S9 and S12 havebeen implicated as biomarkers of inflammation (28-30), no clinicalpancreatitis was observed among the samples used in this study. Lu etal. observed that S100A9 was elevated in pancreatic carcinoma tissuecompared with adjacent control tissue, and proposed that other S100proteins may also serve as markers of pancreatic cancer (31). Recently,Ohuchida et al. extended this finding and showed that S100A6 and S100 μlare also elevated in pancreatic cancer tissue compared with controls andalso in the ductal juices (21, 22). The confidence of identification ofthese S100 homologs A6, A8, A9, and A11 in the cyst fluids in ourcurrent study is very high. No peptides overlapped among the 15 peptidessequenced for the four S100 homologs. S100A8 was detected with 5peptides sequenced and 54% amino acid sequence coverage while homologS100A9 was distinguished using 6 peptides sequenced and 64% coverage.43% sequence coverage was obtained for 3 peptides sequenced for S100A11.Only one peptide of excellent sequence quality and reproducibility wasdetected for S100A6.

Detectable significant levels of CEACAM homologs and S100 homologs inpancreatic cyst fluids, in addition to the presence of mucins and theloss of amylase, are useful biomarkers for the presence or potential ofadenoma and carcinoma. This conclusion is supported by their presence inthe five cysts, #17, 20, 5, 14, 21 that were confirmed by histopathologyand cytopathology to be adenomas or carcinomas, but not at significantlevels in cysts #13, 16, 18, 7, 19, 15, and 1. Cyst 5, anadenocarcinoma, is similar in cyst fluid proteome to non-mucinous cystsexcept for the presence of significant levels of CEACAM1, CEACAM5, andfour 5100 homologs. Cyst 21, a MCA, is similar in cyst fluid proteome tocyst 5 but with less of these proteins. For cyst 19B, a MCA, multiplepotential biomarkers of mucins, CEACAM, and S100 are apparent. Fivemonths earlier, when cyst 19B was aspired as cyst 19A, mass spectrometryhad detected two CEACAMs and multiple mucins, consistent with thesuggestion that CEA of greater than 192 ng/mL may indicate the presenceof mucinous neoplasm (3). Thus S100 homologs are also useful markers ofcyst progression.

Although the high mass accuracy and platform stability of the massspectrometer used in this study facilitated biomarker discovery, oncethe protein names are known, another mass spectrometry method calledMultiple Reaction Monitoring (MRM) can accurately quantify multiplebiomarkers against internal standards at the same time with much highersensitivity (32-35). Importantly, the quantitation of mucin homologs,CEACAM homologs, S100 homologs, amylase, and other marker proteins, canbe performed at the same time using the same method, so theircombination will provide valuable diagnostic and prognostic informationto the clinician. The biomarkers described herein are obtainable fromless than 40 μL of cyst fluids and detection of their presencefacilitates earlier pancreatic cancer detection in cysts than heretoforepreviously possible.

Example 2 Xenograft Model of Pancreatic Cancer and Pancreatic Cyst FluidSecretion

The use of clinical samples for studying the biology of pancreatic cystto cancer is difficult because of inability to obtain time coursematerial in most instances, and because most invasive techniques oflaboratory investigation cannot be used on patients. A mouse model, ifvalid and available, can accelerate pancreatic cancer research. Forexample, mouse stroma cells infiltrating the tumor and supports thetumor growth can be marked with Green Fluorescence Protein by using aGFP transgenic mouse as the host of the xenograft.

In the laboratory of Dr. Repasky, about 33% of pancreatic tumorsengraftment resulted in successful tumor propagation for three passagesfor both adenocarcinomas and neuroendocrine tumors (38, 39). Thesexenograft tissues contain complex cell types from both cancer andstroma. The cancer cells form glands that hold secretions similar towhat is seen in the parent tumors. We obtained three mice for us thatharbored tumors soft to the touch. These mice are normally not used forpreclinical drug treatment experiments. About 500 μL of fluids werecollected from each of these xenograft and we analyzed them using thesame protocol of GelC/MS/MS described in Example 1.

The high mass accuracy and sensitivity of our QSTAR XL mass spectrometerusing LC/MS/MS produced numerous peptide sequences many of which easilydistinguished human homologs of proteins from mouse homologs. Thisability allowed us to interpret whether and how much mouse plasma andstroma are infiltrating the human tumors, and the origin of biomarkerproteins detected in the xenograft fluids. Although these liquid cystsare often believed to result from necrosis, a process called cysticdegeneration of tumors, in our mass spectrometry analysis of threeindependent xenograft fluids, we saw no evidence of general release ofhigh abundance cellular proteins normally happening in necrosis. Similarto the pancreatic cyst fluids that did not contain pancreatic enzymesecretion, the xenograft fluids contained hundreds of plasma infiltrateproteins of mouse origin. However, human proteins were present,including abundant levels of pancreatic cyst fluid neoplasm biomarkerproteins described in Example 1: mucin 1, mucins 5AC, mucin 5B, CEA,CEACAM 6, and S100A6 and S100A11. (Tables 3 and 4). Thus these “cancercells” were secreting the same biomarkers as for the pancreatic cystfluids from patients harboring cystadenoma, IPMN, and adenocarcinoma.Several other proteins in Table 5 below, not assigned as biomarkers inExample 1 are also seen in both cyst fluid and xenograft fluids,indicating that they can be functional biomarkers as well. Validatingthat our cyst fluid biomarkers can be secreted by pancreatic cancerxenograft was exciting, but cyst proteins conspicuously absent wereCEACAM7, S100A8 and A9.

TABLE 5 Table 5. A partial list of proteins found in xenograft fluidsfrom all three separate xenograft experiments. The numbers under eachsample are EMPAI scores roughly proportional to the protein abundance inthe sample. For comparison, serum albumin from mouse plasma infiltrationor blood contamination has an average value of 12. The first 18proteins, in bold, are also found among patient- derived pancreatic cystfluids when cystadenoma, IPMN, or adenocarcinoma were indicated. Title(Bold protein names are also found in pancreatic cyst fluids harboringcystadenoma, emPAI score IPMN, or Xeno- Xeno- Xeno- Swiss-Prot Entryadenocarcinoma but not Mass graft graft graft name in benign cysts) (Da)Sample 1 Sample 2 Sample 3 ANXA2_HUMAN Annexin A2 38808 6.61 9.04 3.38S10A6_HUMAN Protein S100-A6 10230 1.71 0.94 0.94 EZRI_HUMAN Ezrin 694841.42 1.83 0.52 LG3BP_HUMAN Galectin-3-binding 66202 1.03 1.15 0.64protein S10AB_HUMAN Protein S100-A11 11847 0.78 3.23 1.37 GELS_HUMANGelsolin 86043 0.52 0.52 0.13 MOES_HUMAN Moesin 67892 0.45 0.53 0.17ANXA1_HUMAN Annexin A1 38918 0.44 0.58 0.74 PIGR_HUMAN Polymeric- 844290.41 0.9 0.24 immunoglobulin receptor NGAL_HUMAN Neutrophil gelatinase-22745 0.36 3.04 0.86 associated lipocalin ANXA3_HUMAN Annexin A3 365240.34 0.34 0.48 CEAM6_HUMAN Carcinoembryonic 37499 0.33 0.46 0.21antigen-related cell adhesion molecule 6 1433S_HUMAN 14-3-3 proteinsigma 27871 0.29 0.89 0.29 MUC5A_HUMAN Mucin-5AC 135404 0.27 1.01 0.21CEAM5_HUMAN Carcinoembryonic 77489 0.21 0.26 0.15 antigen-related celladhesion molecule 5 MUC1_HUMAN Mucin-1 122170 0.2 0.27 0.16 MUC13_HUMANMucin-13 55710 0.14 0.3 0.21 MUC5B_HUMAN Mucin-5B 605803 0.08 0.16 0.21AGR2_HUMAN Anterior gradient protein 2 20024 1.41 3.09 0.42 ANXA5_HUMANAnnexin A5 35971 1.22 1.7 0.82Other biomarkers of interest include anterior gradient protein 2 whichhas been proposed as a marker of pancreatic cancer tissue because of itsover-expression in most pancreatic cancers (40). Another interestingbiomarkers is NGAL (Neutrophil gelatinase-associated lipocalin), a newearly biomarker of acute kidney injury in rats. Its level in blood riseswithin two hours of renal injury. The protein is a member of the largelipocalin family of extracellular proteins which transports or bindssmall hydrophilic molecules, but when located inside a cell may becomeprotease inhibitors. Its role in pancreatic cyst fluids is may be partlyassociated with inflammation.

Proteomics has resulted in the identification of biomarkers present incysts, a better understanding of the basic biological features of cystsand their natural history, thereby providing a better understanding ofthe molecular profile within these cysts. Such information can be usedto advantage to identify clinically relevant targets for early diagnosisand treatment of pancreatic cancer.

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method of diagnosing an increased risk for the development ofpancreatic cancer in a human test subject, said method comprising: a)obtaining a pancreatic cyst fluid specimen from a human test subject; b)analyzing said fluid specimen for the presence of at least threebiomarkers for pancreatic carcinoma in said cyst fluid sample, saidpresence being indicative of an increased risk for pancreatic cancer,said biomarkers being selected from the group consisting of mucin 1,mucin 2, mucin 5AC, mucin 5B, mucin 6, CEA CAM 1, CEACAM 5, CEACAM 6,CEACAM 7, CEACAM 8, S100-A6, S100-A8, S100-A9 and S100-A11.
 2. Themethod claim 1, wherein said biomarkers comprise at least three mucins.3. The method of claim 1, wherein said biomarkers comprise at least twomucins and at least two CEACAMs.
 4. The method of claim 2 or 3 furthercomprising detection of at least one biomarker selected from the groupconsisting of S100-A6, S100-A8, S100-A9 and S100-A11.
 5. The method ofclaim 1 wherein said diagnosis differentiates a malignant tumor from abenign tumor.
 6. The method of claim 1 wherein said diagnosis is anadjunct to a primary diagnostic test for carcinoma of the pancreas.
 7. Asolid support comprising antibodies immunologically specific for thebiomarkers of claim
 1. 8. The method of claim 1, wherein threebiomarkers are detected, said markers being Mucin 5B, CEACAM 5 andCEACAM
 6. 9. A genetic signature comprising nucleic acids encoding humanproteins indicative of an increased risk of pancreatic cancer,comprising Mucin-5B precursor, Mucin-5AC, Mucin-1 precursor,Carcinoembryonic antigen-related cell adhesion molecule 5 precursor,Carcinoembryonic antigen-related cell adhesion molecule 6 precursor,Tetraspanin-8, Neutrophil gelatinase-associated lipocalin precursor,Anterior gradient protein 2 homolog precursor, Protein S100-A11, andProtein S100-A6.
 10. A panel of biomarker proteins indicative of anincreased risk for pancreatic cancer, affixed to a solid support, saidpanel comprising Mucin-5B precursor, Mucin-5AC, Mucin-1 precursor,Carcinoembryonic antigen-related cell adhesion molecule 5 precursor,Carcinoembryonic antigen-related cell adhesion molecule 6 precursor,Tetraspanin-8, Neutrophil gelatinase-associated lipocalin precursor,Anterior gradient protein 2 homolog precursor, Protein S100-A11, andProtein S100-A6.
 11. The protein panel as claimed in claim 10,consisting of Mucin 5B precursor, Carcinoembryonic antigen-related celladhesion molecule 5 precursor, Carcinoembryonic antigen-related celladhesion molecule 6 precursor, Tetraspanin-8, and Anterior gradientprotein 2 homolog precursor.
 12. A kit for practicing the method ofclaim 1, comprising reagents suitable for detecting the presence orabsence of said at least three biomarkers in said cyst fluid.
 13. A kitcomprising the solid support of claim 7 and reagents effective forassessing immune complex formation between said biomarkers and saidantibodies affixed to said solid support.